The long-term objective of the proposed research is to understand the mechanism of genomic imprinting, a gene regulatory process in mammals that distinguishes the parental origin of alleles. The mechanism is likely to depend on the inheritance of parent-specific methylation patterns, or methylation imprints. Disturbances in the establishment, transmission and perpetuation of these imprints can result in human congenital anomalies. As well, alterations in the imprinting process may contribute to human cancers. The primary objectives of the proposed research are to determine essential requirements for the establishment and transmission of germline methylation imprints in the mouse. These will be addressed in three specific aims. Specific Aim 1: Characterization of genomic requirements for producing a heritable methylation imprint in the maternal germ line. Extensive molecular, genetic and developmental investigations into the regulation of RSVIgmyc transgene imprinting predict that the normally nonimprinted c-myc locus can be imprinted by the targeted introduction of certain sequences. To test the hypothesis that c-myc can be imprinted in this fashion, we propose to introduce sequences into the endogenous c-myc locus via homologous recombination. These experiments are important for understanding the cis-acting features that distinguish an imprinted from a nonimprinted locus. Specific Aim 2: Examination of the role of the Dnmt1 protein in the creation of a methylation imprint in the female germ line. The hypothesis to be tested here is that the protein product of the DNA (cytosine-5)- methyltransferase (Dnmt1) gene is responsible for the establishment of methylation imprints in growing oocytes. The hypothesis will be tested by generating an allele of Dnmt1 in which the oocyte-specific alternative first exon of the oocyte-specific mRNA is deleted (lo allele). These experiments will provide a deeper understanding of the role of Dnmt1 in generating methylation imprints. Specific Aim 3. Determination of the role of the Dnmt1 protein in the inheritance of gametic methylation. A central question will be addressed in this specific aim. Does the protein product of the Dnmt1 gene catalyze the inheritance of methylation imprints during preimplantation development? To address this hypothesis, mice with the mutant lo allele will be evaluated for their ability to maintain (transmit) methylation imprints during preimplantation development.